Livestock plays an important role in economy of Pakistan. Different livestock animals used for for meat, milk, draught, and sports. The genetic data of different cattle breeds like Red Sindhi and Tharparkar is not available which needs to be established for their genetic identification, conservation and to find their genetic diversity among them. Blood samples of pure bred animals were collected from their respective home tracts. The Red Sindhi cattle samples were collected from (Barani Livestock Production Research Institute, Kherimurat, Attock, Govt. Red Sindhi Cattle Breeding Farm, Tando Muhammad Khan and Department of Livestock Management, Sindh Agriculture University, Tandojam) and Tharparkar cattle samples were collected from (Department of Livestock Management, Sindh Agriculture University, Tandojam, Tharparkar cattle Farm at Nabi Sar Road and from Tando Qaiser in Sind). DNA was extracted with the standard protocol in Molecular Biology and Genomics Laboratory of Institute of Biochemistry and Biotechnology. Specific primers were designed by using special softwares Primer 3 for mitochondrial D-loop region and Cytochrom b gene from NCBI accession no. AF492350. Then after primers optimization PCR amplification was done. Then sequencing of target fragments was carried out. Sequences were alligned with the help of software blast2sequence. Single Nucleotide Polymorphisms (SNP5) were identified and comparison of 5 mitochondrial DNA haplotypes of two cattle breeds was done. Sequences were analyzed and compared with already reported sequence of Mitochondrial DNA of Bos indicuss, Bos taurus and Bubalus bubalis available at NCBI.

Phylogenetic tree was constructed using MEGA 4.1 software (http://www.megasoftware.net/MEGA4.1.html) showed that Pakistani, European and Asian cattle are genetically same but different from Buffalo. This work provided the genetic data which is very helpful for determining the genetic diversity of cattle population, breed identification, animal forensic and paternity cases and making effective breeding policies and conservational activities in future. This work is very helpful about breed characterization of two cattle breeds (Red Sindhi and Tharparker) and developing understanding about genetic architecture of cattle breeds as present study conclude that six SNPs were present in both breeds, four private to Red Sindhi and 22 were private to Tharparkar.